Expression Of Acidic Phospholipase A2-1, Gene Cloning And Sequence Analysis Of A Mixed Acidic Phospholipase A2 From Guangxi Ophiophagus Hannah (King Cobra) Venom

A cDNA encoding acidic phospholipase A2-1(APLA2-1) From Guangxi Ophiophagus Hannah(King Cobra) Venom was inserted into bacterial expression vector pBLMVL2, pET28a(+) and expressed in E.coli RR1,BL21 respectively. pBLMVL2-APLA2-1 has no obviously expression. pET28a-APLA2-1 was effectively expressed in E.coli BL21.The protein was produced as insoluble inclusion bodies. After partial purification by washing the inclusion bodies with Triton X-100, denaturing and refolding, the renatured recombinant protein was purified by Sephadex G-75. The expressed protein exhibited enzymatic activity and platelet aggregation inhibiting effect. In addition, synthetic oligonucleotides were used to amplify PLA2 gene by RT-PCR from total RNA of Guangxi Ophiophagus Hannah (King Cobra) gland. The PCR product was cloned into the pMD18-T vector. The positive clones encoding APLA2-1, APLA2-2 and a novel APLA2 which was designed as APLA2-3 were selected. The complete sequences of APLA2-3 were determined by sequencing and its amino acid sequences were deduced. Its isoelectated points, molecular mass, secondary structure and partial physic-chemistry character were predicated by Antheprot 4.3 c software. Its protein sequence showed 64% homology with APLA2-2 and 69% homology with APLA2-1.These would provided a good basis for further discussing the relation of structure and function, and the studying of evolution of PLA2 from snake venom.